The present invention relates to polypeptide molecules that can act as ligands for proteins and other agents that bind platelet and endothelial cell membranes in vivo.
Cells of many types have surface proteins, called "integrins," that are recognized by extracellular proteins, such as fibronectin, vitronectin, ostopontin, collagens, thrombospondin, fibrinogen and von Willebrand factor (VWF), which affect the attachment of cells to their surroundings. Integrins which act as receptors for several of these extracellular proteins have been identified with human platelet glycoprotein Ib (GPIa), which partially mediates VWF-dependent adhesion of platelets to exposed vascular endothelium; GPIIa, which corresponds to the .beta. chain of the fibronectin receptor; and the heterodimer protein complex GPIIb-GPIIIa, which functions in platelets as a receptor for fibrinogen, for VWF in the absence of fibrinogen, for fibronectin and for vitronectin.
An .alpha.-.beta. chain configuration common to GPIIb and GPIIIa is typical of members of the integrin family of receptors. Another group of adhesion-promoting proteins, referred to generically as "cellular adhesion molecules" (CAMs), are classified with proteins encoded by genes of the immunoglobulin gene superfamily. See, e.g., Williams & Barclay, Ann. Rev. Immunol. 6: 381-405 (1988), the contents of which are hereby incorporated by reference. Like other proteins associated with the immunoglobulin (Ig) superfamily, CAMs share a common structure--the immunoglobulin homology unit--that is characterized by an amino-acid sequence, approximately 100 residues in length, having a centrally placed disulfide bridge which stabilizes a series of anti-parallel .beta. strands into the so-called antibody fold. In particular, the immunoglobulin homology unit comprises a conserved amino-acid sequence Gly-X-X-Val/Leu/Ile-X-Val/Leu/Ile-X-Cys/(35-55 amino acids)/Asp-X-Gly-X-Tyr-X-Cys-X-Val/Ala. See Hunkapill-er & Hood, Nature 323: 15 (1986).
Among the known CAMs are intercellular adhesion molecule-1 (ICAM-1), which mediates leukocyte adhesion by binding the integrin lymphocyte function-associated molecule-1 (LFA-1); carcinoembryonic antigen (CEA); fasciclin II; CD4, a T-cell subset marker which is a component of the cellular receptor for HIV-I; neuronal cell adhesion (N-CAM), which mediates adhesion of neural cells; myelin-associated glycoprotein (MAG), which is understood to function in myelination; lymphocyte function-associated antigen-3 (LFA-3) and the major glycoprotein of peripheral myelin (Po). In addition to the functional association of CD4 with HIV-I, the role of CAMs as viral receptors is reflected in the fact that receptors for two picornaviruses, human rhinovirus and poliovirus have been identified, respectively, with ICAM-1 and a cell surface glycoprotein comprised of three immunoglobulin-like loop domains (compared to five such domains for ICAM-1).
Although CAM molecules fulfill important functions in vivo, no protein obtained in substantially pure form from platelets has ever been identified, structurally or functionally, as a member of the CAM family. A characterization of this sort would be significant because of the role(s) such a protein, and variant molecules based thereon, would be expected to play in basic cell surface recognition events involving platelets, including self-association of platelets (aggregation), with other blood cells and with endothelial cells which line blood vessels; and platelet adhesion to extracellular matrix components, for example, subendothelium exposed in the course of vascular trauma.